Oligonucleotide—nanoparticle conjugates have attracted considerable interest because of an example of such conjugates explored by Mirkin and co-workers, who demonstrated a colorimetric DNA detection technique by using oligonucleotide-gold nanoparticle (AuNP) conjugates in the 1990s. The oligonucleotide offers target sequence-specific recognition capability and AuNP imparts solution color change in response to hybridization with the target. Dispersed oligonucleotide-AuNP conjugates appear red due to their characteristic surface plasmon resonance (SPR) absorption band (with absorption peak at ˜520 nm). Upon target hybridization, cross-linking of two sets of conjugates results in particle aggregation and the reduction in interparticle distance causes a red shift of the SPR absorption band, hence the solution color turns purple. Alternatively, a non-cross-link method was designed by Maeda and co-workers that a single set of conjugates aggregated when hybridized with a perfectly complementary target under appropriate salt concentration (i.e., 0.5-2.5 M sodium chloride (NaCl)). Using specially designed sequences (e.g., DNAzyme and aptamer), oligonucleotide-AuNP conjugates have been utilized for the detection of numerous non-nucleic acid analytes, including metal ions, small molecules, proteins, and cells. As another example, oligonucleotide-silver nanoparticle (AgNP) conjugates were also be used for highly sensitive colorimetric detection as its extinction coefficient is larger than that of AuNP. Apart from diagnostics, these oligonucleotide-nanoparticle conjugates, in particular AuNPs, have proved to be very useful for therapeutics (e.g., gene and chemodrug delivery), as well as for the construction of DNA-templated nanostructures.
The most common method of preparing oligonucleotide-AuNP and -AgNP conjugates is by chemisorption of monothiol-modified oligonucleotide onto nanoparticle's surface. However, the chemisorbed oligonucleotide is known to be susceptible to displacement reaction by thiol-containing small molecules (e.g., dithiothreitol (DTT) and mercaptoethanol, frequently used ingredients in enzymatic reaction buffers) as well as to thermal desorption. This stability problem, if not solved, would seriously limit their applications. For example, when a significant portion of the oligonucleotide is desorbed from the AuNP surface, particle aggregation occurs and thus the solution color turns purple even in the absence of any targets. In 0.1 M DTT, this happens within a few minutes. To address this issue, Mirkin and co-workers prepared conjugates with steroid cyclic disulfide and trihexylthiol anchors, which exhibited greatly enhanced stability toward DTT (no aggregation for 2 and 8 hours, respectively). Nevertheless, these oligonucleotides were much more expensive because their syntheses required non-standard phosphoramidites with lower coupling yields. In view of this, Graham and co-workers reported the synthesis of thioctic acid-modified oligonucleotide via treatment of standard 3′-amino-modifier C7 controlled pore glass solid support with N-hydroxysuccinimidyl ester of thioctic acid while Liu and co-workers reported the synthesis of dithiocarbamate-modified oligonucleotide by means of reaction between amino-modified oligonucleotide and carbon disulfide. These two disulfide-linked conjugates had similar stability in DTT as the steroid cyclic disulfide-linked conjugates. Analogous to thiol-gold (S—Au) linkage, thiol-modified oligonucleotide can be conjugated to AgNP via thiol-silver (S—Ag) linkage, but with lower binding affinity. Triple cyclic disulfide and thioctic acid were successfully employed to enhance the stability of oligonucleotide-AgNP conjugates.
Regarding thermal desorption, the S—Au and S—Ag linkages are heat labile. For monothiol-linked oligonucleotide-AuNP conjugates, oligonucleotide desorption occurs readily at a temperature higher than 70° C. This prohibits their utilization in high temperature processes. One example is polymerase chain reaction (PCR), which is the most widely used method to amplify a specific DNA sequence and plays an important role in numerous applications including clinical diagnostics, environmental surveillance, food monitoring, forensic analysis, biowarfare agent detection, as well as biological research. It involves repeated cycling at three temperatures (i.e., 94° C. for template/amplicon denaturation, ˜55° C. for primer annealing, and 72° C. for primer extension). The amount of the specific sequence is doubled at each thermal cycle, hence a single copy of the template ends up in million (20 cycles) to billion (30 cycles) copies of the amplicon. Highly sensitive colorimetric detection of PCR products with oligonucleotide-AuNP conjugates was reported. Nevertheless, post-amplification open-tube addition of the conjugates was unavoidable because they could not withstand the thermal cycling process, which posed a high risk of carryover contamination. Associated with the oligonucleotide desorption during PCR is the non-specific adsorption of Taq DNA polymerase onto the exposed AuNP surface and thus the amplification reaction is inhibited.
In view of the aforementioned stability issues and complicated and expensive solutions available in the art, there is a clear need for a new method of stabilizing oligonucleotide-AuNP and -AgNP conjugates that is easy-to-perform and inexpensive.